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. 2019 Dec 12;5(12):e03012.
doi: 10.1016/j.heliyon.2019.e03012. eCollection 2019 Dec.

Studies on peroxidase production and detection of Sporotrichum thermophile-like catalase-peroxidase gene in a B acillus species isolated from Hogsback forest reserve, South Africa

Ayodeji O Falade  1   2   3 , Leonard V Mabinya  1   2 , Anthony I Okoh  1   2 , Uchechukwu U Nwodo  1   2
Affiliations

Studies on peroxidase production and detection of Sporotrichum thermophile-like catalase-peroxidase gene in a B acillus species isolated from Hogsback forest reserve, South Africa

Ayodeji O Falade et al. Heliyon. .

Abstract

This study sought to determine the process conditions for optimum peroxidase production by a B acillus species (Bacillus sp. FALADE-1-KX640922) isolated from Hogsback forest reserve in South Africa and characterize the peroxidase gene in the bacteria. We optimized peroxidase production by manipulating the environmental and nutritional parameters under submerged fermentation. Subsequently, the gene encoding heme-peroxidase was determined through nested polymerase chain reaction and Sanger DNA sequencing. The studied bacteria had maximum peroxidase production at pH 8, 30 °C and 150 rpm. The addition of guaiacol to lignin fermentation medium enhanced peroxidase production by over 100 % in the studied bacteria. However, the other lignin monomers (veratryl alcohol, vanillin, vanillic acid and ferulic acid) repressed the enzyme activity. Modification of the fermentation medium with ammonium sulphate gave the maximum peroxidase yield (8.87 U mL-1). Under the predetermined culture conditions, Bacillus sp. FALADE-1 expressed maximum specific peroxidase activity at 48 h (8.32 U mg-1). Interestingly, a search of the sequenced gene in PeroxiBase showed 100% similarity to Sporotrichum thermophile catalase-peroxidase gene (katG), as well, the deduced protein sequence clustered with bacterial catalase-peroxidases and had a molecular weight of about 11.45 kDa with 7.01 as the estimated isoelectric point. Subsequently, the nucleotide sequence was deposited in the National Center for Biotechnology Information (NCBI) repository with the accession number MF407314. In conclusion, Bacillus sp. FALADE-1 is a promising candidate for improved peroxidase production.

Keywords: Bifunctional enzyme; Bioinformatics; Biotechnology; Catalase-peroxidase; Enzyme production; Metabolite; Microbiology; Molecular characterization; Optimization; Peroxidase; Proteins.

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Figures

Figure 1
Figure 1
Determination of initial pH for optimum peroxidase production by Bacillus sp. FALADE-1. Each column represent mean ± standard deviation. Error bars with the same alphabet are not significantly different (P > 0.05).
Figure 2
Figure 2
Determination of incubation temperature for optimum peroxidase production by Bacillus sp. FALADE-1. Each column represent mean ± standard deviation. Error bars with the same alphabet are not significantly different (P > 0.05).
Figure 3
Figure 3
Determination of agitation rate for optimum peroxidase production by Bacillus sp. FALADE-1. Each column represent mean ± standard deviation. Error bars with the same alphabet are not significantly different (P > 0.05).
Figure 4
Figure 4
Effect of lignin model compounds on peroxidase production by Bacillus sp. FALADE-1. Each column represent mean ± standard deviation. LGO: lignin only (control), LG + GA: lignin and guaiacol, LG + VALC: lignin and veratryl alcohol, LG + VAN: lignin and vanillin, LG + VA: lignin and vanillic acid, LG + FA: lignin and ferulic acid. Error bars with different alphabets are significantly different (P < 0.05).
Figure 5
Figure 5
Effect of nitrogen supplementation on peroxidase production by Bacillus sp. FALADE-1. Each column represent mean ± standard deviation. YEO: yeast extract only (control), YE + AN: yeast extract and ammonium nitrate, YE + AC: yeast extract and ammonium chloride, YE + AS: yeast extract and ammonium sulphate. Error bars with different alphabets are significantly different (P < 0.05).
Figure 6
Figure 6
Growth pattern and kinetics of peroxidase production by Bacillus sp. FALADE-1.
Figure 7
Figure 7
Phylogenetic tree showing the family of Bacillus sp. FALADE-1 peroxidase in the bacterial heme-peroxidases from PeroxiBase. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Red tips indicate Catalase-peroxidase, black tips indicate DyP-type Peroxidase while the blue tips represent Di-heme Cytochrome C Peroxidase (DiHCcP). The red triangular tip indicates the studied protein sequence (BAFPrx). The UniProtKB reference numbers/GenBank accession number* of the proteins are indicated in parentheses. Afla: Anoxybacillus flavithermus, Ame: Alkaliphilus metalliredigenes, BAspN: Bacillus sp. NRRL B-14911, Bbr: Brevibacillus brevis, Bha: Bacillus halodurans, Gsp: Geobacillus sp., Aaeo: Aquifex aeolicus, Ks: Kuenenia stuttgartiensis, Mmag: Magnetospirillum magneticum, Rp: Rhodopseudomonas palustris, Rsph: Rhodobacter sphaeroides, Sth: Symbiobacterium thermophilum, Eco: Escherichia coli, Pf: Pseudomonas fluorescens, Sbo: Shigella boydii, EcoH7: Escherichia coli 0157:H7, BAFPrx: Bacillus sp. FALADE-1 Peroxidase, KatG: Catalase-peroxide, DyPPrx: Dye Decolourizing Peroxidase.
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